The ability to conveniently separate small or large quantities of materials from mixtures with high purity and yield is crucial to many disciplines, including chemistry, biochemistry, molecular biology, immunology and cell biology, where the separation and isolation of inorganic and organic molecules and biological materials such as enzymes, nucleic acids, cells and organelles are often required.
Conventional methods for the separation of biological materials include liquid chromatography, using supports such as silica gel or agarose, gas chromatography, analytical and preparative centrifugation, electrophoresis and the like. These methods, however, are time consuming, and may require complicated, multistep procedures and complex instrumentation. Furthermore, these techniques do not readily provide aseptic materials, making the usage of the recovered materials unsuitable for therapeutic use, such as in cell transplantation. Additionally, the lengthy procedures as conventionally practiced reduce the recovery of viable cells and the stability of labile molecules.
Another method for these separations utilizes magnetic particles that are coated with msbps (members of a specific binding pair) either directly (for example U.S. Pat. Nos. 3,970,518 and 4,018,886 to Giaever) or through the intermediacy of coating materials that allow the attachment of msbps (for example, U.S. Pat. Nos. 4,230,685 to Senyei, 4,452,773 to Molday, 4,554,088 to Whitehead, 4,267,235 to Rembaum, and 4,157,323 to Yen). The msbps are chosen to have affinity for the material to be separated. Thus, by taking advantage of the magnetic properties of the particles, the materials are separated in a magnetic field (Giaever, U.S. Pat. No. 3,970,518).
Magnetic separations are superior to other separation technologies because they are rapid, non-toxic, require only simple devices and may easily be performed under sterile conditions.
Magnetic particles are most useful in separations if they meet certain criteria. They should be easily separated in a magnetic field. They should remain in uniform suspension for significant periods of time. It should be easy to attach msbps to them. There should be very little non-specific binding to the particles. The particles should be easily redispersed after the magnetic separation to facilitate the recovery of the purified material.
A wide variety of magnetic particles for use in separations have been prepared by workers in the field. The methods used to prepare the magnetic particles can be roughly divided into two types. The first method involves the coating of an existing magnetic material. The second method involves the generation of the magnetic material in the presence of the coating material. Magnetite is the most common magnetic material used, since some particles prepared from freshly precipitated magnetite are claimed to be superparamagnetic (see, for example, U.S. Pat. No. 4,827,945 to Groman), a property which facilitates resuspension of the particles after magnetic separation. Some of the patents mention the use of other magnetic materials as well. Representative examples of the first method are Giaever '518 (proteins adsorbed onto nickel microspheres), U.S. Pat. No. 4,554,088 to Whitehead (functionalized polymeric silane coating on magnetite), U.S. Pat. Nos. 5,512,332 and 5,597,531 to Liberti (adsorption of serum albumin onto aggregates of magnetite or other magnetic metal oxides during or immediately after ultrasonic disruption of the aggregates), U.S. Pat. No. 4,157,323 to Yen and U.S. Pat. Nos. 4,358,388 to Daniel (polymerization of monomers in the presence of magnetite), and 4,230,685 to Senyei (adsorption of Protein A to magnetite).
Representative examples of the second method are U.S. Pat. No. 4,452,773 to Molday (precipitation of magnetite in the presence of dextran), U.S. Pat. No. 4,795,698 to Owen (precipitation of magnetite in the presence of serum albumin), and U.S. Pat. No. 5,262,176 to Palmacci (precipitation of magnetite in the presence of arabinogalactan). A related method is the precipitation of magnetite in the pores of, or on the surface of, an existing particle, as, for example in U.S. Pat. No. 4,654,267 to Ugelstad.
The range of particle sizes available is generally limited by the method used to make the particles. Precipitation of magnetite in the presence of a coating material generally produces particles under about 50–60 nm in diameter. Coating of existing magnetic particles generally yields particles greater than 500 nm in diameter, since particles of magnetite and most other magnetic material are generally heavily aggregated under the coating conditions used. The existing particles under 50–60 nm stay suspended almost indefinitely, but can only be separated in specialized magnetic devices. Particles greater than 500 nm in diameter are easily separated magnetically, but settle out at an inconvenient rate. Particles between 50 and 500 nm can be prepared and coated (Liberti '332 and '531) but the process used is both inconvenient and potentially injurious to the coating material. Thus there is a great need for a convenient preparation of magnetic particles for use in magnetic separations which properly combine facile magnetic separation and very slow settling rate.
Various means have been used to attach msbps to the magnetic particles. As noted above, this can be done either by direct adsorption of the msbps to the magnetic particles or by attachment of the msbps to a coating which has been placed on the magnetic particle. Proteins directly adsorbed to surfaces typically lose some of their biological activity, so most workers prefer to attach msbps to a coating that has been placed on the particle. A variety of coating materials have been used which provide a means of attaching msbps either covalently or through a strong specific binding interaction. Representative examples are described in Senyei '685 (Protein A, which allows attachment of immunoglobulins), Liberti '332 (biotinylated serum albumin, which allows attachment of avidin or streptavidin), Whitehead '088 (polymerized aminosilane, providing amino groups for covalently attachment of msbps), Rembaum '235 (polyglutaraldehyde for attachment of msbps with available amino groups), Molday '773 (dextran, which is further modified to provide aldehyde and amino groups for covalent attachment of msbps), U.S. Pat. No. 5,411,863 to Miltenyi (dextran modified with cyanogen bromide for attachment of msbps with amino or thiol groups) and U.S. Pat. No. 5,512,439 to Homes (monodisperse magnetic polystyrene beads with surface tosyloxy groups to which amine-modified nucleic acids can be attached).
In order for magnetic particles to stay in suspension for significant periods of time, they must not only have the appropriate size initially, but must also be colloidally stable, that is, they must not grow in size by aggregation. A variety of methods have been used for preparing colloidally stable magnetic particles. The two usual methods for maintaining the colloidal stability of particles are to impart a high surface charge or to coat the particle with a hydrophilic polymer. Representative examples of these two methods can be found in U.S. Pat. No. 4,329,241 to Massart (iron oxide-based magnetic fluids stabilized by the surface charge on the iron oxide surface itself), U.S. Pat. No. 4,208,294 to Khalafalla (surfactant-stabilized magnetic particles), U.S. Pat. No. 4,935,147 to Ullman (magnetic particles coated with succinylated BSA to maintain the surface charge at neutral pH), and Molday '773, Palmacci '176, and U.S. Pat. No. 4,101,435 to Hasegawa (hydrophilic polymers such as the polysaccharides dextran and arabinogalactan).
Of the coating materials which have been used, it will be noted that polysaccharides have been used as coating materials both to impart colloidal stability to the magnetic particles and to provide a means of attaching msbps to the magnetic particles. Thus, polysaccharides, and in particular dextran, appear to be particularly useful coating materials. In order to be useful, though, the msbps must be stably attached to the magnetic particle. This means that the msbps must be stably attached to the polysaccharide coating, and the polysaccharide coating must in turn be stably attached to the magnetic particle. The methods described in the literature for attaching msbps to polysaccharides, in particular, dextran, are not satisfactory. Molday '773 uses periodate oxidation of the dextran as means of introducing functional groups onto the particle surface to which msbps can be attached. Periodate oxidation, however, is known to weaken the dextran chain, and fission of the dextran chains can lead to loss of attached msbps. Miltenyi '863 describes the modification of the dextran coating with cyanogen bromide to allow attachment of msbps. It is well known from work on attachment of msbps to affinity chromatography supports that cyanogen bromide provides a labile linkage to the msbps. Thus, although polysaccharides, and dextran in particular, would be preferred coating materials for magnetic particles, polysaccharide-coated magnetic particles with stable linkages to the attached msbps have not been reported.
The stability of polysaccharide coatings attached directly to magnetic particles has not been studied extensively. When magnetite is precipitated in the presence of dextran (Molday '773) or carboxymethyldextran (U.S. Pat. No. 5,204,457 to Maruno) the attachment of the dextran to the magnetic particle appears to be stable. Simple incubation of either dextran or carboxydextran with colloidal magnetic iron oxide particles at room temperature, however, results in a labile coating. Incubation at elevated temperatures instead improves the stability, but Maruno '457 notes that carboxymethyldextran-coated magnetic particles prepared in this way have poorer stability than those prepared by precipitating magnetite in the presence of the carboxymethyldextran. As noted above, however, particles prepared by precipitation of magnetite in the presence of a coating material are typically smaller than 50–60 nm, and thus are difficult to separate magnetically. Thus, a means of stably attaching the polysaccharide coating to the magnetic particle other than incubation at elevated temperature may be needed.
Maruno '457 describes the preparation of magnetic particles coated with carboxyalkylethers of dextran, although the use of the carboxyl group for attachment of msbps is not anticipated. Attachment of msbps to a surface coated with carboxymethyldextran has been reported in B. Johnnson, et al., Analytical Biochemistry 198, 268 (1991). The use of carboxyalkylethers, however, is not satisfactory. Carboxymethyldextran readily forms a lactone between the carboxyl group and a hydroxy group on one of the anhydroglucose units of the dextran chain (N. D. Heindel, et. al., Bioconjugate Chem, 5, 98 (1994), suggesting that this hydroxyl group can catalyze the hydrolysis of any linkage between an msbps and the carboxymethyl group. The carboxyethyl groups of carboxyethyldextran can be lost via a retro-Michael reaction. Carboxyalkylethers with longer alkyl groups are expected to render the polysaccharide coating hydrophobic, leading to unacceptable non-specific binding to the particle surface. Introduction of carboxyl functionality into a polysaccharide as a means of attaching msbps would be useful. The methods which have been previously described may be adequate for some purposes, but there is still a need for a method of preparing a hydrophilic polysaccharide with pendant carboxyl groups to which msbps can be stably attached.
Accordingly, there remains a need for a convenient method for obtaining coated magnetic particles that have the following characteristics. The particles should be as small as possible in order to form stable colloidal solutions but large enough to have a significant magnetic moment, and thus be rapidly and easily separable with a small magnet. The particles must also have a large surface area to allow the binding of an amount of the material of interest in order to eliminate multi-step procedures to recover the requisite amount of material.
The coating of the aggregates also determines utility. The coating material should be stably associated with the iron core, and allow stable covalent bond formation between the coating material and the coupling molecule to allow firm attachment. It should be resistant to the non-specific binding of undesired materials present in the solution. It is preferable that the coating is non-toxic, sterilizable and preferably biodegradable to allow transplantation of the separated material directly into an animal or human patient.
An additional desirable characteristic of the magnetic particles is that they should only minimally affect the physical properties of the separated material. For example, the identity of cell populations separated with magnetic particles is conveniently analyzed by flow cytometry. Flow cytometry uses physical parameters to determine the types of cells isolated and for accurate analysis, these physical parameters must be only minimally affected by attached magnetic particles.